Aspirin Interacts with Cholesterol-Containing Membranes in a pH-Dependent Manner

Aspirin has been used for broad therapeutic treatment, including secondary prevention of cardiovascular disease associated with increased cholesterol levels. Aspirin and other nonsteroidal anti-inflammatory drugs have been shown to interact with lipid membranes and change their biophysical properties. In this study, mixed lipid model bilayers made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) comprising varying concentrations of cholesterol (10:1, 4:1, and 1:1 mole ratio of lipid:chol), prepared by the droplet interface bilayer method, were used to examine the effects of aspirin at various pH on transbilayer water permeability. The presence of aspirin increases the water permeability of POPC bilayers in a concentration-dependent manner, with a greater magnitude of increase at pH 3 compared to pH 7. In the presence of cholesterol, aspirin is similarly shown to increase water permeability; however, the extent of the increase depends on both the concentration of cholesterol and the pH, with the least pronounced enhancement in water permeability at high cholesterol levels at pH 7. A fusion of data from differential scanning calorimetry, confocal Raman microspectrophotometry, and interfacial tensiometric measurements demonstrates that aspirin can promote significant thermal, structural, and interfacial property perturbations in the mixed-lipid POPC or DOPC membranes containing cholesterol, indicating a disordering effect on the lipid membranes. Our findings suggest that aspirin fluidizes phosphocholine membranes in both cholesterol-free and cholesterol-enriched states and that the overall effect is greater when aspirin is in a neutral state. These results confer a deeper comprehension of the divergent effects of aspirin on biological membranes having heterogeneous compositions, under varying physiological pH and different cholesterol compositions, with implications for a better understanding of the gastrointestinal toxicity induced by the long term use of this important nonsteroidal anti-inflammatory molecule.


List of Supplemental Figures
(A) Raman spectra of POPC and aspirin mixture (POPC to aspirin of 1 to 1 mole ratio at pH 3) in the CH stretching region, the aspirin spectra are scaled to the intensity of the 1606 cm -1 , the solid green line shows the spectrum of POPC after subtraction of the aspirin spectrum (solid orange line) from the original spectrum (dotted green line), (B) superposition of spectra of POPC and aspirin lipid mixtures in the Raman shift region between 2800 and 3000 cm -1 after subtraction.S6.
List of Supplemental Tables Table S1.Effect of aspirin on osmotic water permeability (µm/s) at 30℃ for POPC and mixed POPC:chol bilayer at pH 3 and pH 7.
Table S2.Effect of aspirin on osmotic water permeability (µm/s) at 30C for DOPC and mixed DOPC:chol bilayer at pH 3 and pH 7.
Table S3.Thermodynamic parameters (Tm and H) for main phase transition of DOPC MLVs at different concentration of aspirin at pH 3 and pH 7.
Table S4.Thermodynamic parameters (Tm and H) for main phase transition of mixed DOPC:chol MLVs at different concentration of aspirin at pH 3 and pH 7.
Table S8.Interfacial parameters for the water/DOPC/SqE and water/DOPC:chol/SqE interfaces in the presence of ASA at pH 7, and 25 ℃ 1. Water permeability analysis using model membrane formed by the droplet interface bilayer (DIB) method The water permeability measurement was performed using the model membrane formed by the droplet interface bilayer (DIB) method.A DIB is formed when aqueous microdroplets bounded by lipid monolayers create a region that has a structure essentially the same as the double-leaflet lipid bilayer of cell membranes (Figure S1A).When two osmotically unbalanced microdroplets were made to adhere at a bilayer, the osmotic gradient drives water transport through the droplet bilayer (the direction of water transport is shown with the arrow in Figure S1B), resulting in a visible change in droplet diameter.Any electrolyte flux is expected to be negligible compared to that of water, as ion permeation is typically almost eight orders of magnitude slower than that of water.The corresponding changes in droplet volume over time (dV/dt) is measured optically by microscopic observation; and the behavior of the system follows the expression of equation ( 1) based on Fick's Law: where A is the geometric bilayer area, νw is the molar volume of water (18 mL/mol), ΔC(t) is the osmolality gradient between two droplets, and  is the bilayer permeability coefficient of water.The volume change with time (dV/dt) is related to the bilayer permeability coefficient of water,  , as expressed in the Equation (1).When the bilayer contact area is constant, the time evolution of the swelling droplet can be obtained from the following equation derived from the integration of eqn. 1, with the following simplifying assumption: since one of the droplets (the shrinking droplet) contains no osmotic agent, its concentration does not change with time: 1, 2 + 1 (2) Using the measured values for: initial size of the osmotic (swelling) droplet; bilayer contact area (A); and initial osmolarity of the osmotic droplet (Co), then the coefficient  for bilayer water permeability may be derived from eqn. 2 from the slope of the curve obtained by plotting (V/Vo) 2 as a function of time.All data points presented in this paper are an average (n ≥ 30) of individual permeability runs, each of which took place over a time course (∼5 min) for osmotic water movement across the droplet bilayer, during which time the droplet contact area (A) remains constant.The recorded videos and images were post-analyzed to measure the dimension of droplets and contact area using custom built image analysis software.All droplet pairs had substantially the same initial size relative to each other, in the diameter range of 100 ± 5 μm diameter.

Contact Angle Measurement
For the contact angle () measurement, two apposing iso-osmotic droplets are made to contact with each other.From the microscopic video images of the two adherent droplets, the contact angle can be measured by considering the geometry of the contacting spheres (as given in eqn. 3) based on geometrical parameters shown in Figure S2, 0] Reported values are from the average of 10 or more measurements.

Water permeability coefficients
The osmotic water permeability coefficients ( ) of mixed bilayers of POPC:chol at 30°C as a function of varying mole fraction of aspirin, at both pH 3 and pH 7 are shown in Table S1.

Thermotropic property
The thermodynamic data, Tm and H, from endothermic DSC thermograms for mixed DOPC:chol MLVs in the presence of different concentrations of aspirin at pH 3 are shown in Table S2 and S3.

Structural property
Figure S4 shows the Raman spectra of POPC lipid bilayer (top) and aspirin (bottom) at room temperature.The detailed characteristic peak assignments for POPC are shown in Table S4.The CH stretching region (2800  3000 cm -1 ) also has peaks from aspirin that interfere with peaks from POPC.Therefore, appropriate subtraction of aspirin peak is necessary to monitor the effect of aspirin on the packing properties of POPC hydrocarbon chains.Before spectral subtraction, the aspirin spectra were scaled to the intensity of the 1606 cm -1 (aromatic C=C stretching from aspirin).The resulting hydrocarbon chain contribution from POPC is shown in green solid line in Figure S4A, along with before (dotted) subtraction of the scaled aspirin spectrum (solid orange).
The spectra shown in Figure S4B are those obtained after the spectral subtraction, allowing for the elimination of the aspirin components for varying mole fraction of aspirin in POPC.S7.
A B A B

Figure S1.
Figure S1.Schematics of (A) aqueous microdroplets surrounded by self-assembled structures, for use as a biomembrane model, and (B) typical DIB-based osmotic water permeability measurement Figure S1.Schematics of (A) aqueous microdroplets surrounded by self-assembled structures, for use as a biomembrane model, and (B) typical DIB-based osmotic water permeability measurement

Figure S3 .
Figure S3.The relative percentage change (%) in osmotic water permeability ( / , where  represents the osmotic water permeability in the absence of ASA) of DOPC and mixed bilayer formed from DOPC:chol (1:1 mole ratio) at 30℃ with varying mole fraction of ASA, at (A) pH 3 and (B) pH 7.

Figure S4 .
Figure S4.Raman spectra of pure POPC lipid bilayer (film of POPC liposome) and aspirin (dried film) at room temperature.

Figure S5.
Figure S5.(A) Raman spectra of POPC and aspirin mixture (POPC to aspirin of 1 to 1 mole ratio at pH 3) in the CH stretching region, the aspirin spectra are scaled to the intensity of the 1606 cm -1 , the solid green line shows the spectrum of POPC after subtraction of the aspirin spectrum (solid orange line) from the original spectrum (dotted green line), (B) superposition of

Figure S2 .
Figure S2.A microscopic picture of two adherent droplets in SqE in the presence of DOPC 5 mM.R1 and R2 are the radii of the respective two droplets and r is the radius of the contact zone between the droplets, and the contact angle () is determined by the eqn.3. The scale bar on the image represents 100 μm.(Adapted from Wood, Megan, Michael Morales, Elizabeth Miller, Samuel Braziel, Joseph Giancaspro, Patrick Scollan, Juan Rosario, Alyssa Gayapa, Michael Krmic, and Sunghee Lee."Ibuprofen and the phosphatidylcholine bilayer: membrane water permeability in the presence and absence of cholesterol."Langmuir 37, no. 15 (2021): 4468-4480.Copyright 2021 American Chemical Society)

Figure S3 .
Figure S3.The relative percentage change (%) in osmotic water permeability ( / , where  represents the osmotic water permeability in the absence of ASA) of DOPC and mixed bilayer formed from DOPC:chol (1:1 mole ratio) at 30℃ with varying mole fraction of ASA, at (A) pH 3 and (B) pH 7.

Figure S4 .
Figure S4.Raman spectra of pure POPC lipid bilayer (film of POPC liposome) and aspirin (dried film) at room temperature.

Figure S5 .
Figure S5.(A) Raman spectra of POPC and aspirin mixture (POPC to aspirin of 1 to 1 mole ratio at pH 3) in the CH stretching region, the aspirin spectra are scaled to the intensity of the 1606 cm -1 , the solid green line shows the spectrum of POPC after subtraction of the aspirin spectrum (solid orange line) from the original spectrum (dotted green line), (B) superposition of spectra of POPC and aspirin lipid mixtures in the Raman shift region between 2800 and 3000 cm -1 after subtraction.

Table S1 .
Effect of aspirin on osmotic water permeability (µm/s) at 30℃ for POPC and mixed POPC:chol bilayer at pH 3 and pH 7.* * Each data represents an average of individual permeability runs (n>30 independent samples) and standard deviation as error bars.

Table S2 .
Effect of aspirin on osmotic water permeability (µm/s) at 30C for DOPC and mixed DOPC:chol bilayer at pH 3 and pH 7.

Table S4 .
Thermodynamic parameters (Tm and H) for main phase transition of DOPC MLVs at different concentration of aspirin at pH 3 and pH 7. Thermodynamic parameters (Tm and H) for main phase transition of mixed DOPC:chol MLVs at different concentration of aspirin at pH 3 and pH 7.

Table S5 .
Peak assignments of POPC Raman spectra 3

Table S8 .
Interfacial parameters for the water/DOPC/SqE and water/DOPC:chol/SqE interfaces in the presence of ASA at pH 7, and 25 ℃ * Each data represents the average for 38 independent samples. *